Sensing chemical-induced DNA damage using CRISPR/Cas9-mediated gene-deletion yeast-reporter strains

Abstract Microorganism-based genotoxicity assessments are vital for evaluating potential chemical-induced DNA damage. In this study, we developed both chromosomally integrated and single-copy plasmid–based reporter assays in budding yeast using a RNR3 promoter–driven luciferase gene. These assays were designed to compare the response to genotoxic chemicals with a pre-established multicopy plasmid–based assay. Despite exhibiting the lowest luciferase activity, the chromosomally integrated reporter assay showed the highest fold induction (i.e., the ratio of luciferase activity in the presence and absence of the chemical) compared with the established plasmid-based assay. Using CRISPR/Cas9 technology, we generated mutants with single- or double-gene deletions, affecting major DNA repair pathways or cell permeability. This enabled us to evaluate reporter gene responses to genotoxicants in a single-copy plasmid–based assay. Elevated background activities were observed in several mutants, such as mag1Δ cells, even without exposure to chemicals. However, substantial luciferase induction was detected in single-deletion mutants following exposure to specific chemicals, including mag1Δ, mms2Δ, and rad59Δ cells treated with methyl methanesulfonate; rad59Δ cells exposed to camptothecin; and mms2Δ and rad10Δ cells treated with mitomycin C (MMC) and cisplatin (CDDP). Notably, mms2Δ/rad10Δ cells treated with MMC or CDDP exhibited significantly enhanced luciferase induction compared with the parent single-deletion mutants, suggesting that postreplication and for nucleotide excision repair processes predominantly contribute to repairing DNA crosslinks. Overall, our findings demonstrate the utility of yeast-based reporter assays employing strains with multiple-deletion mutations in DNA repair genes. These assays serve as valuable tools for investigating DNA repair mechanisms and assessing chemical-induced DNA damage. Key points • Responses to genotoxic chemicals were investigated in three types of reporter yeast. • Yeast strains with single- and double-deletions of DNA repair genes were tested. • Two DNA repair pathways predominantly contributed to DNA crosslink repair in yeast. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-024-13020-w.

Luminescence intensity derived from luciferase activity (a-e) and fold induction (f-j) are shown with standard deviations.S6).S7).Bar plots with asterisks indicate the gene-deletion mutant cells with significant luminescent intensity compared with that of the corresponding wild-type cells (Student's t-test, p < 0.01; Table S10).

Fig. S1 .
Fig. S1.Luciferase induction with five genotoxic chemicals in the BY4741 strain carrying a chromosomally integrated and a multicopy plasmid with a RNR3 promoter-driven luciferase gene.

Fig. S2 .
Fig. S2.Level of luciferase activity without chemicals in the wild-type and seven DNA repair gene-deleted strains carrying a single-copy RNR3 promoter-driven luciferase reporter plasmid.

Fig. S4 .
Fig. S4.Luciferase induction with HU in three gene-deletion mutants defective in cell permeability.

Table S3 . Primer sets for confirmation of deleted allele by colony PCR Target
3-218R 435Repair DNAs for CRISPR/Cas9-mediated gene disruption were prepared by connecting 5'-and 3'-flanking DNAs by PCR.Target genes, primer sets for preparation of both flanking DNAs for target gene and repair DNA sizes are shown.genes, primer sets for confirmation of deleted target genes by colony PCR are shown with the expected sizes of colony PCR products from the wild-type and deleted alleles.

Table S4 . Statistical analysis by Student's t -test for three reporter systems
Statistical significance of luciferase activity was tested by a two-tailed paired Student's t -test between with and without HU in each assay.P -values <0.01 are indicated in bold.Statistical significance of luciferase activity was tested by t -test between assay systems.P -values <0.01 are indicated in bold.-test (a two-tailed paired) using luciferase activities and fold inductions fromwild-type cells and the correponding gene-deletion mutant treated with indicated concentration of each chemical are shown in each table.Statistically significant p -values (<0.01) are indicated in bold.

Table S7 . Statistical analysis by t-test of luciferase activities and fold inductions in rad59 D -derived double-gene deletion mutants treated with camptothecin (CPT)
Significance of luciferase activities and fold inductions tested by Student's t -test between rad59 D cells and the rad59 D -derived double-gene deletion mutant treated with indicated concentration of CPT.Statistically significant p -values (<0.01) are indicated in bold.

Table S8 . Statistical analysis by Student's t-test of luciferase activitives and fold inductions in mms2 D -derived double-gene deletion mutants treated with mitomycin C (MMC) Statistical
significance of luciferase activities and fold inductions tested by Student's ttest between mms2 D cells and the mms2 D -derived double-gene deletion mutant treated with indicated concentration of MMC.Statiscally significant p -values (<0.01) are indicated in bold.

Table S9 . Statistical analysis by t-test of luciferase activities and fold inductions in rad10 D and mms2 D -derived double-gene deletion mutants treated with cis-dichlorodiammine platinum (CDDP)
Statistical significance of luciferase activities and fold inductions tested by Student's ttest between rad10 D and the rad10 D -derived double gene deletion mutant (upper panel), and mms2 D and mms2 D -derived double-gene mutant (lower panel) cells treated with indicated concentration of CDDP.Statistically significant p -values (<0.01) are indicated in bold.

Table S10 . Statistical analysis by t-testof luciferase activities and fold inductions in pdr1 D , pdr3 D , and erg6 D -cells treated with hydroxyurea (HU)
Statistical significance of luciferase activityies and fold inductions tested by Student's t -test between the wild-type cells and indicated gene-deletion strain.Statistically significant p -values (<0.01) are indicated in bold.